Glutamine Metabolism Promotes Renal Fibrosis through Regulation of Mitochondrial Energy Generation and Mitochondrial Fission

Fibroblast activation and proliferation is an essential phase in the progression of renal fibrosis. Despite the recognized significance of glutamine metabolism in cellular growth and proliferation, its precise pathophysiological relevance in renal fibrosis remains uncertain. Therefore, this study aims to investigate the involvement of glutamine metabolism in fibroblast activation and its possible mechanism. Our findings highlight the importance of glutamine metabolism in fibroblast activation and reveal that patients with severe fibrosis exhibit elevated serum glutamine levels and increased expression of kidney glutamine synthetase. Furthermore, the deprivation of glutamine metabolism in vitro and in vivo could inhibit fibroblast activation, thereby ameliorating renal fibrosis. It was also detected that glutamine metabolism is crucial for maintaining mitochondrial function and morphology. These effects may partially depend on the metabolic intermediate α-ketoglutaric acid. Moreover, glutamine deprivation led to upregulated mitochondrial fission in fibroblasts and the activation of the mammalian target of rapamycin / mitochondrial fission process 1 / dynamin-related protein 1 pathway. Thus, these results provide compelling evidence that the modulation of glutamine metabolism initiates the regulation of mitochondrial function, thereby facilitating the progression of renal fibrosis. Consequently, targeting glutamine metabolism emerges as a novel and promising avenue for therapeutic intervention and prevention of renal fibrosis.


Animals and unilateral ureteral obstruction
Unilateral ureteral obstruction (UUO)-induced mouse renal interstitial fibrosis was constructed according to the reported method.Firstly, animals were anesthetized with 1% sodium pentobarbital by i.p. injection.The unilateral dorsal incision was made, then the left ureter was exposed and ligated.All mice were euthanized on the 14 th day after surgery and the kidneys were fixed in 4% paraformaldehyde for further analysis simultaneously.All procedures of animails were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, and conducted according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health (NIH), Bethesda, MD, USA).
Male C57BL/6 mice (8 weeks old, weighing 200-25g) were obtained from Beijing Weitong Lihua Experimental Animal Company (Beijing, China).Animals were bred and maintained in the Tongji Medical College of Huazhong University of Science and Technology.The mice were randomly and equally allocated into four distinct groups: the sham group, the sham + BPTES group, the UUO group, and the UUO + BPTES group.Each group consisted of three mice.The allocation was performed by a technician who was not involved in the research, using a table of random numbers.The degree of fibrosis was evaluated through the assessment of fibrotic tissue using histological staining and the quantitative analysis of fibrosis-related markers.
The extent of fibrosis was assessed by histopathological staining and examination of fibrosis-specific markers.

Histological analysis.
Kidneys were fixed in 4% paraformaldehyde and then embedded in paraffin.The 4 μm renal sections were stained to assess the renal tubular damage.Damaged tubules were defined based on tubular dilation, brush border loss, necrosis, and cast formation.
Masson and Sirus Red staining were performed to determine the degree of renal interstitial fibrosis.Measurement of the fibrotic area was quantified with ImageJ software (NIH).

Immunohistochemistry and immunofluorescence staining
Paraffinized renal sections were deparaffinized in xylene and hydrated using consecutive ethanol washes of 100%, 95%, 75%, and 50%, and antigen retrieval was performed using citrate solution.The endogenous peroxidase was blocked with 3% H2O2 for 15 minutes, non-specific proteins were blocked with 10% goat serum for 30min.Then, the sections were incubated with primary antibodies of GLS1 (Proteintch, USA), Fibronectin (Proteintch, USA), Collagen I (Proteintch, USA), α-SMA (Abcam, USA) at 4℃ overnight.For immunohistochemistry, the slides were then incubated with HRP-conjugated secondary antibody at room temperature for 30 min and visualized with diaminobenzidine substrate.The nucleus was counterstained with hematoxylin, and the slides were visualized under a light microscope.The sections were exposed to Cy3-labeled secondary antibodies for immunofluorescence and were observed with a fluorescence microscope after staining the nucleus with DAPI.

Human samples
Serum

Cell culture
The rat kidney interstitial fibroblast cell lines (NRK-49F) were purchased from American Type Culture Collection (Manassas, USA).The cells were routinely cultured in Dulbecco's Modified Eagle's Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37℃ under 5% CO2.Cells that reached approximately 60% confluence were used for in vitro experiments.For the transforming growth factor (TGF)-β1-induced fibrosis model, cells were incubated in a serum-free culture medium for 24 h before exposure to 10 ng/ml recombinant human TGF-β1 (Peprotech, USA) for 24 h.

Cell transfection
For small interfering RNA (siRNA) experiments, GLS siRNA or scrambled siRNA were designed and synthesized.Cells were further transfected with siGLS according to experimental design.Infection efficiency was estimated by western blot.For transfection, cells were seeded in 6-well plates, grown overnight until 60-70% confluent, and then transfected with 50 nM (final concentration) GLS siRNA.Six hours after transfection, the cells were treated with 10 ng/ml TGF-β1.

Untargeted metabolomics
Non-targeted global metabolomic profiles were generated through Appiled Protein Technology (Shanghai) by employing ultra-performance liquid chromatography coupled with high resolution/accurate mass spectrometer (UPLC-MS/MS).To detect a comprehensive range of metabolites, four platforms were utilized: 1) UPLC-MS/MS with positive ionization, 2) UPLC-MS/MS with negative ionization, 3) UPLC-MS/MS polar platform with negative ionization, and 4) gas chromatography-MS.Metabolites were identified based on their m/z retention time and by comparing them to library entities of purified known standards.
Metabolites were analyzed using electrospray ionization (ESI) in auto MS/MS positive scan mode, with a range of 20-1,300 m/z.The ESI source utilized dry nitrogen gas at a flow rate of 10 L/min and a drying temperature of 220˚C.The ESI capillary voltage was set at 4,500 V with a nebulizer pressure of 2.2 bar.For MS2 acquisition, the collision energy was set at 20 eV and the end Plate Offset at 500 V. Separation of metabolites was achieved using a Hamilton® Intensity Solo 2 C18 column (100 mm × 2.1 mm × 1.8 μm), with sodium formate used as the calibrant for external calibration.
The gradient program employed a flow rate of 0.250 ml/min, utilizing a composition of 99A:1.0Bfrom 0.00-2.00min, transitioning to a composition of 99A:1.0B to 1.0A:99B from 2.00-17.00min, maintaining a composition of 1.0A:99B from 17.00-20.00min, and then transitioning back to a composition of 1.0A:99B to 99A:1.0B from 20.00-20.10min.Subsequently, the flow rate was adjusted to 0.350 ml/min, maintaining a composition of 99A:1.0Bfrom 20.10-28.50min.Finally, the flow rate returned to 0.250 ml/min, with a composition of 99A:1.0Bfrom 28.50-30 min, resulting in a total run time of 30 min.The maximum pressure reached during the process was 14993 pounds per square inch (PSI).The autosampler temperature was set at 8˚C, while the column oven temperature was maintained at 35˚C.A total volume of 10 μl was injected into the QTOF MS.

Seahorse Energy Metabolism Instrument
The XFe-96 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent, Santa Clara, CA, USA) was utilized to conduct the measurements.NRK-49F cells were seeded into Seahorse tissue culture plates (Seahorse XF96 V3 PS Cell Culture Microplates #101085-004) at a density of 2000 cells per well.Prior to loading into the XF-96 apparatus, the cells were incubated in a CO2-free incubator to achieve equilibration.

Figure S2 (
Figure S2 and renal biopsy samples were obtained from patients diagnosed with IgA nephropathy, displaying varying degrees of renal fibrosis.Samples were provided by the Department of Pathology, Tongji Hospital of Huazhong University of Science and Technology.The studies involving human kidney sections and serum were conducted with the informed consent of the patients and were approved by the Institutional Ethics Committee at Tongji Hospital of Huazhong University of Science and Technology.Renal pathologists, who were unaware of the patients' clinical data, assessed interstitial inflammation and interstitial fibrosis (mild, moderate, or severe) in accordance with established pathology guidelines.